RNA interference (RNAi) is a gene silencing phenomenon mediated by double-stranded RNA (dsRNA) molecules, shutting down homologous gene expression at mRNA level. RNAi is a post-transcriptional gene regulation mechanism, also named post-transcriptional gene silencing (PTGS), target gene knockdown, or gene silencing.
RNAi phenomenon was first reported in plants by two different research groups in 1990. Later on, this phenomenon was further observed in almost all eukaryotes, including C. elegans, Drosophila, zebrafish and mice.
In 1999, RNA fragments of 21 to 25 nucleotides in length were identified in plant RNAi by Hamilton and Baulcombe. These small RNA fragments were demonstrated to be the mediator necessary for RNAi, and thus named as small interfering RNA (siRNA).
Double-stranded siRNA conjugates with endogenous enzymes and proteins, and then forms RNA-induced silencing complex (RISC). In the process of RNAi, while the sense RNA strand of double-stranded siRNA is excluded from the complex, the antisense RNA strand functions to guide RISC to target mRNA at homologous locus, resulting in the degradation of target mRNA and gene silencing mediated by RNase III component within RISC complex.
However, due to the vulnerability of siRNA to serum ribonuclease that resulting in serum instability, synthetic siRNAs are often chemically modified so as to increase their serum stability and gene silencing activity.
Even though, in consideration of the poor understanding of the degradation process and mechanism of RNAi, random modification strategy is performed to stabilize siRNA in body fluids. Although this strategy can improve serum stability of the modified siRNAs to some extent, it usually ends up with excessive chemical modifications and results in increased cytotoxicity and compromised biological activity in many cases, due to the lack of theoretical guidance. This therefore restricts the in vivo applications of the modified siRNAs. In addition, the extensive siRNA modification strategy also limits the use of many chemical modifications that have great stabilizing effect, however with relatively high cytotoxicity.
Therefore, there is an urgent need to develop a specific modification strategy for synthetic siRNAs, to achieve optimum serum stability with minimal chemical modifications.